The purpose of this investigation is to extend studies demostrating the effects of the protein, glutamine synthetase (GS), on the visible spectrum of the dye Cibacron Blue F3GA. Four chromatographically distinct subfractions of Cibacron Blue have been isolated. The difference spectra obtained when each of these subfractions binds to taut, relaxed, or subunit forms of GS clearly differ from each other and can be displaced by addition of ADP to the dye plus enzyme sample. Incubation of one of the dye subfractions with GS results in an altered difference spectrum with time which can no longer be displaced by ADP. Time-dependent changes in the visible dye spectrum are obtained upon dissociation of the protein in urea with differences seen for GS 2.6 and GS 11. These changes can be correlated with light scattering data obtained under the same conditions. An aromatic spectral perturbation is obtained in the standard urea dissociating system. A method is described for quantitating aromatic amino acid residues in intact denatured proteins using second derivative UV spectroscopy.